Single-cell analysis of mast cell degranulation induced by airway smooth muscle-secreted chemokines

Abstract

Background: Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma.

Methods: Carbon-fiber microelectrode amperometry was used to study the effects of ASM-secreted chemokines on mouse peritoneal MC degranulation.

Results: MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin release per granule was correlated with increased spike half-width and rise-time values.

Conclusions: MCs are directly activated by ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery.

General significance: The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation.

Keywords: Asthma; Carbon-fiber microelectrode amperometry; Exocytosis.

Figure 1

Representative CFMA traces from MCs incubated with 0.5 μg ml⁻¹ anti-TNP (with the exception of the A23187-stimulation without IgE condition) IgE and stimulated via calcium ionophore (10 μM A23187), IgE-mediated activation (200 ng ml⁻¹ TNP-OVA), or ASM-secreted cytokines (200 ng/ml CXCL10 or CCL5).

Figure 2

Total serotonin released from MCs stimulated with A23187 without IgE pre-incubation (n=18), A23187 with IgE pre-incubation (n=17), TNP-OVA (n=19), CXCL10 (n=26), or CCL5 (n=16). Total serotonin is calculated using the average Q value per spike and the number of spikes recorded for a given CFMA trace. * indicates p<0.05 versus TNP-OVA condition. “n” refers to the number of traces that were collected.

Figure 3

Average spike area (A) and spike frequency (B) values for MCs stimulated with A23187 without IgE pre-incubation (n=18), A23187 with IgE pre-incubation (n=17), TNP-OVA (n=19), CXCL10 (n=26), or CCL5 (n=16). * indicates p<0.05 versus TNP-OVA condition. “n” refers to the number of traces collected.

Figure 4

Average spike half-width (A) and spike rise-time (B) values for MCs stimulated with A23187 without IgE pre-incubation (n=18), A23187 with IgE pre-incubation (n=17), TNP-OVA (n=19), CXCL10 (n=26), or CCL5 (n=16). * indicates p<0.05 versus TNP-OVA condition. “n” refers to the number of traces collected.